This invention relates to a novel bioactive polypeptide useful as a carcinostatic and a polydeoxyribonucleic acid (hereinafter referred to as DNA) having genetic information thereof.
This invention also relates to a replicable recombinant DNA containing the above DNA and a microorganism or a cell transformed with the replicable recombinant DNA, and further relates to the novel bioactive polypeptide obtained by expression of the DNA's genetic information and a process for producing the same.
Lymphotoxin (hereinafter referred to as LT) is a protein having antitumor activity derived from lymphocytes, which is expected to have clinical application in a manner similar to tumor necrosis factor (hereinafter referred to as TNF) derived from macrophages. These proteins, LT and TNF have been obtained by adding an endotoxin or a phorbol ester to lymphocytes or macrophages, respectively, for activation thereof. However, with the progress of gene manipulation techniques, the structures of these proteins have recently become identified. As a result, it has become possible to produce these proteins by cultivation of microorganisms or cells using the genes coding for these proteins. Gray et al. [Nature, 312, 721 (1984)] and Pennica et al. [Nature, 312, 724 (1984)] each succeeded in cloning them using the gene manipulation techniques.
On the other hand, through advances in gene techniques, the mass-production of LT and TNF has recently become possible so that their clinical trials can be conducted. However, the results of clinical tests, particularly the clinical data of TNF, are not necessarily satisfactory [T. Taguchi Cancer and Chemotherapy, 13, 3491 (1986), I. Urushizaki, Oncologia, 20, 105 (1987) and A. Klansne, Biotech., 5, 335 (1987)]. The most serious problem encountered is the appearance of strong side effects. The administration of TNF is accompanied by fervescence, ague, trepidatio, depression of blood pressure and the like. Therefore, TNF has a disadvantage that the growth of tumors can not be prevented due to the necessity of the interruption of its administration. In addition, the tumor selectivity of TNF is not as good as was first thought. It has been found that normal cells and fibroblasts also have TNF receptors [Y. Niitsu, Therapeu. Res., 7, 275 (1987)], and normal cells which react with TNF have also been discovered.
The clinical results with respect to LT are not as plentiful as those about TNF. However, from the fact that LT has a structure very similar to that of TNF and has the property of combining with common receptors on cells [B. Y. Rubin, et al. J. Exp. Med., 162, 1099 (1985)], it is presumed that the nonspecific cytotoxicity to non-tumor organs, tissues and cells will likewise appear and side effects similar to those of TNF will be induced.
Thus, conventional TNF and LT present a complicated clinical picture. Therefore the application of novel preparations, including new modifications and combination therapy are desired.
On the other hand, as drugs for killing tumor cells selectively, antitumor immune conjugates, each of which is prepared by combining an anti-cancer antibody with a chemotherapeutics or a biotic toxin, have been developed. These have a characteristic of recognizing tumor-specific antigens or tumor-associated antigens on tumor cells, combining therewith and terminating the DNA synthesis or the protein synthesis of the tumor cells to kill them. Therefore, these drugs are specific against tumor organs, tissues and cells, and have little side effect to normal cells. Some antibody-medicament or antibody-biotic toxin conjugates have already been submitted to clinical applications, and several good results have been obtained.
In order to enhance the tumor specificity of LT which is a human-derived toxin having strong cytotoxic activity, the present inventors have studies to obtain a novel immunotoxin combined with an antitumor antibody, under the technical background described above. In the preparation of such an immunotoxin, two important problems ave ben considered. One is that the conventional LT or LT mutein has the high possibility of combining with an antibody at an active site on the molecule and the biological activity of LT itself is impaired in the course of the above combining. The other is that the expression of the activity of LT si presumed to be very little, because the toxin portion, namely the LT molecule, is not liberated from the antibody after incorporation of LT into tumor cells and accordingly difficult to transfer to a target site in the cells, even if the immunotoxin is synthesized somewhat maintaining the biological activity of LT.
In this specification, amino acids and peptides are indicated by the abbreviations adopted by IUPAC-IUB Committee of Biochemistry Nomenclature (CBN). For example, the following abbreviations are used:
Gln: Glutamine residue PA0 Asp: Asparatic acid residue PA0 Pro: Proline residue PA0 Tyr: Tyrosine residue PA0 Val: Valine residue PA0 Lys: Lysine residue PA0 Glu: Glutamic acid residue PA0 Ala: Alanine residue PA0 Asn: Asparagine residue PA0 Leu: Leucine residue PA0 Phe: Phenylalanine residue PA0 Gly: Glycine residue PA0 His: Histidine residue PA0 Ser: Serine residue PA0 Thr: Threon PA0 Ile: Isoleucine residue PA0 Trp: Tryptophan residue PA0 Arg: Arginine residue PA0 Met: Methionine residue PA0 Cys: Cystine residue PA0 A: 2'-Deoxyadenylic acid residue PA0 C: 2'-Deoxycytidylic acid residue PA0 G: 2'-Deoxyguanylic acid residue PA0 T: Thymidylic acid residue
When the optical isomer is capable of existing with respect to the amino acids and the like, the L-form is represented unless otherwise specified.
Also in this specification, polymers or oligomers of DNA are indicated by the sequence of the following abbreviations:
Unless otherwise stated, the direction from the left to the right in sequence indicates the direction from the 5'-position to the 3'-position.